Percoll plus gradient

Colonic surgical specimens were collected and processed immediately after surgical resection, as previously described [20] . Briefly, specimens were opened longitudinally, rinsed and examined for macroscopic morphological alterations, and representative full-thickness samples were obtained. Intestinal mucosa was stripped from the muscularis mucosa, cut into strips and washed in Hank's Balanced Salt Solution (HBSS; Invitrogen/GIBCO) containing 10 mM DTT (Sigma-Aldrich) in order to remove mucus. IEC were isolated by repeated incubations in HBSS containing 1 mM EDTA (Sigma-Aldrich). A 40% Percoll Plus gradient (GE Healthcare), where the IEC was equilibrated at the interface, was used in order to remove mononuclear, red blood and dead cells; resulting preparations enriched for IEC were collected and washed twice in PBS, and subsequently counted. Cell preparations were stained by means of an immunoperoxidase method, with an anti-keratin mAb and a monoclonal antibody directed against a leukocyte common Ag (CD45), resulting in a <1% contamination of epithelial cell preparations by LPMC.